Chronic non-healing wounds represent a major health care buriden especially in elderly, bed-ridden, and diabetic patient populations. Microbial bioburden and/or infection are generally acknowledged to deleteriously affect wound healing and are a significant source of medical complications. Little is known about the microbes that colonize chronic wounds and how they influence the host cutaneous defense response during wound healing. Most microbial surveys of chronic wounds have relied on culture-based methodologies, even though it is estimated that >99% of bacteria are estimated to resist isolation in pure culture. Sequencing bacterial 16S ribosomal RNA (rRNA) genes is a less-biased approach to characterizing bacterial diversity. 16s rRNA genes are present in every bacterial cell and can be used to identify bacteria. Using these methods, I have generated preliminary data in the Lepr^'' (db/db) mouse model of impaired wound healing that demonstrated a longitudinal selective shift in wound microbiota coinciding with impaired healing. Concurrent transcriptional profiling showed prolonged expression of cutaneous defense response genes, correlating with the selective shift observed in wound microbiota. Based on this preliminary data, the specific aims of this proposal test the hypothesis that wound microbiota integrates with an aberrant cutaneous host defense response to impair wound healing: (1) Characterize role for microbes in impaired wound healing by manipulating host skin and/or wound microbiota using gnotobiotic (germ-free) mice and antibiotic treatment regiments and (2) Investigate the impact of the cutaneous defense response on host microbiota and wound healing using db/db mice deficient for innate immune components or pro-inflammatory innate immune molecules. The completion of these aims will provide new insight into microbial interactions with the cutaneous defense response in impaired wound healing. The long-term goal is to enable the development of improved biomarkers and novel therapeutics by leveraging our understanding of host-microbe interactions in impaired wound healing. Furthermore, implementation of the proposed career development plan and completion of these aims will establish the foundation necessary for me to embark on a career as an independent investigator.